www.nature.com/cddis ARTICLE OPEN Puma, noxa, p53, and p63 differentially mediate stress pathway induced apoptosis 1 1 2 1 3 1 1 Jun Wang , Holly R. Thomas , Zhang Li , Nan✉ Cher (Florence) Yeo , Hannah E. Scott , Nghi Dang , Mohammed Iqbal Hossain , Shaida A. Andrabi1,4 and John M. Parant 1 © The Author(s) 2021 Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/ puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele. Cell Death and Disease (2021) 12:659 ; https://doi.org/10.1038/s41419-021-03902-6 INTRODUCTION doxorubicin-induced neural cell death in mouse embryos [35]; Cellular stress response occurs when homeostasis is perturbed [1]. however not in irradiated mouse thymocytes [36]. This suggests Prolonged acute stress or chromic stress often results in cell death tissue-specificinfluences occur. In addition, the transcription to remove the stressed cell from the organism. The apoptotic factor p63 can regulate the intrinsic apoptosis in response to ER response to stress is often pathological and associated with stress through mediating Puma expression [19, 37, 38]. p73 can human diseases [2–8]. Among stress pathways, DNA damage also mediate ROS stress to increase the BAK/BCL-2 ratio in human stress, unfolded protein stress, and oxidative stress responses cells [39]. These studies suggest not only p53, but also p63 and have been linked to multiple human pathologies and can be p73 have the potential to mediate multiple stress-induced distinguished by distinct proximal signaling components but can apoptotic outcomes through the induction of the pro-apoptotic also converge downstream on the p53 family of stress sensors and Bcl-2 family members, such as PUMA and NOXA [40–42]. However, the apoptotic signaling network [9–17]. Interestingly, the cellular which of the p53 family and/or proapoptotic family are essential response to a stress can also be cell/tissues dependent [18–23]. for the different stress responses is unclear. Deeper understanding of consequences of cellular stress and Zebrafish is a useful model to understand the in vivo pathology mediators of stress pathways in vivo will facilitate avenues to associated with human diseases. We and others have demon- mediate disease pathogenesis. strated zebrafish are a model of cancer predisposition, heart The p53 family (tp53, tp63, and tp73) acts as mediators of development, neurodegeneration, and many others [43–47]. Many apoptotic stress response [24–27]. The tumor suppressor p53 is of diseases and stress pathways are conserved and ~82% of activated by a number of cellular stressed including but not human disease genes have zebrafish homologs [48]. For example, limited to genotoxic stress, ribosomal stress, and oncogenic stress in the p53 pathway p53, p63, and p73; the negative regulators [28–34]. Further, p63 and p73 have been shown to be required for mdm2 and mdm4; the downstream transcriptional targets, such as 1Department of Pharmacology and Toxicology, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA. 2Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, USA. 3Department of Biology, University of Alabama at Birmingham Collage of Arts and Sciences Department and Genetics Department, University of Alabama at Birmingham School of Medicine, Birmingham, AL, USA. 4Department of Neurology, University of Alabama at ✉ Birmingham School of Medicine, Birmingham, AL, USA. email: [email protected] Edited by B. Zhivotovsky Received: 21 January 2021 Revised: 27 May 2021 Accepted: 28 May 2021 Official journal of CDDpress J. Wang et al. 2 p21, puma, noxa, cyclin G, and gadd45a are conserved in 1:1 orthology. Here, we took advantage of the properties of zebrafish embryos to analyze apoptotic outcomes in response to genotoxic stress, ER stress, and oxidative stress in a number of genetic null animals. We generated six knockout alleles including puma/bbc3, noxa/pmaip1, p53, mdm2, p63, and p73 with multiple genome- editing techniques. Utilizing these mutants, we defined: (1) that the apoptosis response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. (2) The ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. And (3) the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. RESULTS Multiple cellular stresses induce transcriptional induction of puma and noxa in zebrafish To first determine if pro-apoptotic mRNAs were upregulated in zebrafish following diverse cellular stresses, we analyzed the relative expression of puma, noxa, bax, and bid (Fig. S1 depicts zebrafish orthology analysis) in 24 hours post fertilization (hpf) embryos exposed to either the genotoxic stress (30 Gy ionizing radiation, IR), ER stress (5 μM Thapsigargin, Thaps.), or oxidative stress (3.3 μM Phorbol 12-myristate 13-acetate, PMA). As with human cells [49, 50], puma has the strongest induction following IR, then noxa, followed 1234567890();,: by bax and bid have mild to no induction (Fig. 1A). Similar to IR, following Thaps. and PMA, both puma and noxa were significantly upregulated, however bax and bid were not induced (Fig. 1B, C). Together these data have indicated that puma and noxa are strongly transcriptionally regulated by cellular stresses. While the p53 family of stress mediators are largely controlled at the post- translational level, we analyzed the relative expression of p53, p63, Fig. 1 Quantitative real-time PCR (qRT-PCR) analysis of pro- apoptotic markers after IR- and drug-induction in wild-type and p73 after these stresses (Fig. S2). Only after IR did we observe fi fi increases in p53 mRNA, which is self-inducing (Fig. S3). p73 was zebra sh embryos. 24 hpf zebra sh embryos were treated with A 30 Gy IR-irradiation, B 5 μM Thapsigargin (Thaps.), and C 3.3 μM significantly induced after IR (this induction is p53 dependent—Fig. fi Phorbol 12-myristate 13-acetate (PMA); and qRT-PCR was performed S3), non-signi cantly induced with Thapsigargin treatment, and at 6 h (A)or4h(B, C) after treatment. Expression levels were significantly reduced after PMA treatment. However, p63 mRNA was normalized to GAPDH. n = 9(A, B) and n = 7(C) from ~30 pooled not significantly induced by any of the treatments. embryos per sample. Bars represent mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Fold change (FC) is indicated. Generation of zebrafish null mutants To further pursue the importance of puma and noxa in the stress- induced apoptotic response in zebrafish, we generated zebrafish others have indicated that IR-induced apoptosis primarily occurs puma and noxa null alleles (Figs. 2, S4 and S5). PUMA and NOXA in the neural tube of 24 hpf zebrafish embryos and is p53 have been described to be transcriptionally induced in a p53 dependent [43, 59–61]. This is consistent with mouse studies dependent as well as p63 and p73 dependent manner [19, 35– demonstrating apoptosis predominantly in the neural tube of 13.5 39, 51–53]. Therefore, to further evaluate the stress pathways we dpc embryos after IR treatment [62]. To determine if puma and have also generated a p53 null allele (Figs. 2 and S6), as well as noxa are required for the p53 dependent, as well as if p63 or p73 p63 and p73 null alleles (Figs. 2, S7 and S8) in zebrafish. To contribute to the apoptotic response in zebrafish, we treated − − − − − − − − introduce an alternative mechanism of p53 induction and zebrafish wild type, tp53 / , tp63 / , tp73 / , bbc3 / , and − − evaluate our new p53 null allele and puma null allele, we also pmaip1 / embryos with 30 Gy IR-irradiation and stained for the generated a mdm2 null allele (Figs. 2 and S9). MDM2 is E3 apoptotic marker activated Caspase-3 at 1 hour post irradiation Ubiquitin ligase. Mouse and zebrafish genetic experiments have (hpi), 6 hpi, and 24 hpi. In wild-type embryos, we do not observe established that deletion of the negative regulator of p53 [54, 55], apoptosis within 1 h, but do by 6 hpi and this persists into 24 hpi MDM2, results in embryonic lethality due to unregulated (Figs.